Multidisciplinary studies with mutated HIV-1 capsid proteins reveal structural mechanisms of lattice stabilization

HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.


Supplementary Figure 18. Van der Waals inter-hexameric interaction energies.
Distributions of Van der Waals interaction energies of inter-hexamer interactions.The analysis considered the interaction energies of sets of atoms, situated at inter-hexameric interfaces and identified via distance cutoff, over which the statistics shown above were computed.For wild type, n=6,151 atoms; for P38A, n=4,074 atoms; for P38AT16I, n=4,585 atoms; for E45A a , n=3,362 atoms; for E45A b , n=4,287 atoms; for E45AR132T, n=3,597 atoms.Supplementary Table 1

Supplementary Tables
The spectra of all the mutants are very similar to that of WT, demonstrating conservation of the overall global fold.The effects of the P38A may result from subtle changes in the overall structure and from its involvement in the intra- A204, L205, G206, P207, G208, M215, I216, A217, Q219, G220, V221 mutation directly relieves electrostatic repulsion, resulting in stabilization of the E45A CA hexamer and the core.In the P38A mutant, the network of interactions around E45 is altered, together with a network of additional residues over 3 neighboring CA subunits (Fig. 1), leading to the "loosening" likely causing the observed destabilization of the P38A CA hexamers and the core.R132T and P216I, are able to partially offset the effect of primary mutations.R132T partially restores the overall net charge of CA, while T216I stabilizes inter-hexamer interactions.ND -no data * -these studies show contradictory results ** -the infectivity of P38A and E45A mutant viruses are significantly impaired Supplementary Table 2

Figure 1 .Supplementary Figure 3 .Supplementary Figure 6 .Supplementary Figure 7 .Supplementary Figure 8 .Supplementary Figure 9 .
Cartoon schematic of mature HIV-1 CA and related nomenclature.Full hexagons represent mature CA hexamers assembled by six CA monomers.Left represents hexamer-hexamer interactions at the 3-fold interface, labeled as CA_hex1, CA_hex2, and CA_hex3 and colored as grey, navy, and purple, respectively.Top right represents a single hexamer (CA_hex1) with three adjacent CA monomers, CA", CA, CA', represented as triangles and colored in cyan, red, and brown, respectively.Bottom right shows a model of the wild-type CA hexamer structure (PDB ID: 4XFX) with colors corresponding to the above cartoon.N-terminal domains (CANTDs) are shown in light hues, C-terminal domains (CACTDs) are shown in dark hues.The effects of mutations on polar and water-mediated contacts around residue 45 and the salt bridge between P1 and D51.(a) Orientation of CA WT hexamer with the side view of one representative monomer (white) and its interaction with the adjacent subunit (grey) outlined in dashed line and enlarged.Locations of mutation sites P38 (blue), T216 (pink), E45 (orange), and R132 (green) are shown as spheres.The effects of mutations on the region around residue 45 and the salt bridge between P1 and D51 (enlarged views of the boxed region, solid line) are shown in (b-g).Polar and water-mediated contacts in CA WT (b), P38A (c), P38A/T216I (d), E45A a (e), E45A b (f), and E45A/R132T (g).Black dashed lines indicate atoms within 3.6 Å. Waters are shown as red spheres.Selected side chains are shown explicitly and labeled.Surface representation of respective views are colored according to electrostatic potential from −10 kBT/e (red) to +10 kBT/e (blue).Supplementary Figure 4. Structural changes associated with P38A/T216I mutations.(a) A CA hexamer is shown in surface view representation with three neighboring intrahexamer CA monomers colored in orange (subunit ΄), yellow (subunit without prime symbol), and green (subunit ΄΄); the other three are shown in gray.Select mutation sites in neighboring subunits are marked with red (A38) and white (I216΄΄) stars.Regions likely affected by the P38A mutation are shown in light blue surface; residues likely affected by the T216I΄΄ mutation are shown in magenta surface.The regions in red, black, and blue boxes are depicted in (b), (c), and (d), respectively.(b-d) Superposition of WT (cartoon ribbons of three neighboring subunits colored in green, yellow, and orange) and P38A/T216I (in pink) CA.Mutations alter CANTD-CANTD (b, c) and CANTD-CACTD interfaces (d).Specific residues affected by P38A and T216I mutations (in red) are shown as sticks.Dashed lines are shown between residues that are within 4 Å.For clarity, residues G220΄΄, A204΄΄ are not shown.Box colors in (b-d) correspond to the boxed regions in panel (a).Dashed box in (b) is an insert of a region within the other box in (b).Supplementary Figure 5. E45A can cause rearrangements at inter-hexamer interfaces, while addition of the R132T mutation reverses these changes in E45A/R132T.(a) Two neighboring hexamers of E45A a CA are shown in surface view.Least squares superposition (alignment based on residues 17-143) of E45A a (dark brown and dark purple CACTDs) with WT (light orange and light pink CACTDs) and E45A/R132T (orange and magenta CACTDs) structures.(b) Enlarged view of the boxed region in (a) shows changes in the position of helices α9, 310, α10, and α11 in both hexamers (marked as hex1 and hex2).The blue arrow indicates the distance between α9_hex1 and α9_hex2 in E45A a , while the black arrow indicates the distance between α9_hex1 and α9_hex2 in WT and E45A/R132T.Addition of the R132T mutation reverses the effect of the E45A mutation at these interfaces.Crystal structures of native WTCPSF6 and WTNup153.(a) CPSF6 and Nup153 peptides bind at the PF74 binding pocket, which is between the CANTD of one CA monomer (no prime) and the CACTD of a neighboring CA monomer within a hexamer (denoted by prime symbols).Enlarged views show the details of how CPSF6 (blue sticks) and Nup153 (green sticks) bind at the PF74 binding pocket.Fo-Fc maps at σ=2.5 are shown in green.Peptide labels are italicized and colored in red.(b) Comparison of native WTCPSF6 (blue) vs. cross-linked CA in complex with CPSF6 (CAXL-CPSF6, yellow, left panel) and native WTNup153 (green) vs. cross-linked CA in complex with Nup153 (CAXL-Nup153, orange, right panel) demonstrates significant changes at the 2-fold interhexamer interface.(c) Comparison of native WTCPSF6 (blue) vs. cross-linked CA in complex with CPSF6 (CAXL-CPSF6, yellow, left panel) and WTNup153 (green) vs. cross-linked CA in complex with Nup153 (CAXL-Nup153, orange, right panel) demonstrates significant changes at the 3-fold inter-hexamer interface.(d) Comparison of native WT CA (WTCA, gray) vs. native WTCPSF6 (blue, left panel) and native WTCA (gray) vs. native WTNup153 (green, right panel) reveal subtle changes at the 2-fold inter-hexamer interface.(E) Comparison of native WTCA (gray) vs. native WTCPSF6 (blue, left panel) and native WTCA (gray) vs. native WTNup153 (green, right panel) reveal subtle changes at the 3-fold interhexamer interface.Conformational changes caused by E45A affect access to the PF74/CPSF6/Nup153 binding pocket.(a) Least squares superposition (alignment based on residues 17-145) of E45A a (green CANTDs, purple CACTDs) with WT CA (CA), E45A/E132T CA, WT CA in complex with a CPSF6 peptide (WTCPSF6), WT CA in complex with a Nup153 peptide (WTNup153), WT CA in complex with PF74 (WTPF74), (gray CANTDs, light pink CACTDs).Two intra-hexamer CA monomers are shown (neighboring subunit is marked by a prime symbol).(b) Enlarged view of the boxed region in (a) showing the entrance to the PF74/CPSF6/Nup153 binding pocket.The change in position of helix α9 in the neighboring subunit (marked with a prime symbol) between E45A a (dark purple CACTD) and the other structures (light pink CACTDs) is noted with a black arrow.The red explosion graphic denotes regions of steric clash between the CPSF6 peptide and PF74 with α9' of E45A a CA.Effects of capsid mutations on assembly.(a-b) Cryo-EM analysis of CA mutant assemblies.Projection images were recorded at low (a) and high (b) magnifications from the corresponding samples as indicated.Scale bars, 1 µm in (a), and 100 nm in (b), respectively.(c) Pelleting assay for CA mutant assemblies.Four CA mutants and CA WT are labeled.'S' and 'P' stand for the supernatant and pellet from each sample.Protein products are visualized by Coomassie Blue staining.Molecular weight markers are labeled on the right.Experiments were performed as three biological replicates, with representative experiments shown above.An image of the uncropped gel in (c) is shown in the Source Data file and below on page 42.Effects of capsid mutations on HIV-1 core stability.An in vitro HIV-1 core stability assay was performed using INsfGFP (green) and CypA-DsRed (red) labeled pseudoviruses immobilized on poly-L-lysine coated coverslips and permeabilized by brief exposure to saponin (see Methods).(a, c) Images showing CypA-DsRed puncta immediately before (top panel) and 25 min or 5 min after (bottom panel) virus membrane permeabilization with saponin (SAP).(b, d) The kinetics of CypA-DsRed loss from INsfGFP-labeled HIV-1 cores over time at room temperature.Arrows in (b) and (d) mark the time of CsA (5 µM) addition at 25 min post-permeabilization to displace CypA-DsRed from remaining HIV-1 cores.Plots are means and standard errors from 4 independent experiments; for each experiment, 4 fields of view were analyzed.Scale bar in (a, c) is 2 µm.Supplementary Figure 13.Ions and water transfer rates of CA hexamers.(a) Chloride ion transfer rates of hexamers.(b) Sodium ion transfer rates of hexamers.(c) Water transfer rates of hexamers.For all panels and for each construct denoted on the xaxis, rates were computed from intervals of 1,000 frames, yielding n=12 inward and outward rate measurements per simulation, over which means and standard errors were computed.Supplementary Figure 14.C-alpha RMSF of CA WT and mutants.(a) RMSF of CA WT, E45A a , E45A b , and E45A/R132T mutants.(b) RMSF of CA WT, P38A, and P38A/T216I mutants.Short dash lines represent ± standard deviation.The lines below represent the sequence positions of key secondary structure elements (helices, loops) in CA WT.Helices in CANTD: helix α1 (residues 17 to 30), helix α2 (36 to 43), helix α3 (49 to 57), helix α4 (63 to 83), helix α5 (101 to 104), helix α6 (111 to 119) and helix α7 (126 to 145), are in gray; helices in CACTD: helix α8 (161 to 173), helix α9 (179 to 192), helix α10 (196 to 205) and helix α11 (211 to 217), are in orange; β-hairpin (1 to 13) is in black, the purple line stands for CypA-binding loop (residues 85 to 93) and the 310 helix (150 to 152) is in orange.(c) Ca RMSD of CA hexamers.

Figure 19 .
Crystal structures of CA mutants and WT / CPSF6 or Nup153 peptides.X-ray crystal structures of WT CA / CPSF6 peptide (a), WT CA / Nup153 peptide (b), and P38A (c), P38A/T216I (d), E45A a (e), E45A b (f), E45A/R132T (g) CAs.Left panel shows the asymmetric unit (asu), a CA monomer, which assembles into the hexameric biological assembly (middle two panels, side and top views).Structures in the first three panels are represented in surface view.The right panel shows electron density (2Fo-Fc maps shown in blue mesh contoured at σ=1.0) around the bound peptides in (a-b) or mutations of interest in (c-g).Colors as noted in figure; light gray surface represents symmetry-related N-terminal domains (CANTD) in the biological assembly, while dark gray surface represents the symmetry-related C-terminal domains (CACTD) in the biological assembly.Some mutations are obstructed from view in the specific orientations above.

Table 3 . Interface area, solvation energy gain, and binding energy calculated for various CA structures.
Å 2 -Interface Area defined as the half sum of the buried surface area 2 ΔG, kcal/mol -Solvation Energy gain 3 BE, kcal/mol -Binding Energy Supplementary